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Regular Manuscript

Activation of Hypoxic Response in Human Embryonic Stem Cell-Derived Embryoid Bodies

¹ Univ of Minnesota
² University of Minnesota

^* To whom correspondence should be addressed. E-mail: kaufm020{at}umn.edu.

	Abstract

Oxygen tension can provide an important determinant for differentiation and development of many cells and tissues. Genetic regulation of hemato-endothelial commitment is known to respond to oxygen deprivation via stimulation of hypoxia inducible factors (HIFs). Here, we use a closed bioreactor system to monitor and control the dissolved oxygen during differentiation of human embryonic stem cells (hESCs) via formation of embryoid bodies (hEBs). Exposing hESC-derived EBs to ambient oxygen at or below 5% results in stabilization of HIF-1 and increased transcription of hypoxic responsive genes. Interestingly, we find that rather than HIF-1 expression being stable over prolonged (7-16 days) culture in hypoxic conditions, HIF-1 expression peaks after approximately 48 hours of hypoxic exposure, and then declines to near undetectable levels, despite constant hypoxic exposure. This transient stabilization of HIF-1 during hESC-derived EB culture is demonstrated for four distinct stages of differentiation. Furthermore, we demonstrate hEB-cell expansion is slowed by hypoxic exposure, with increased apoptosis. However, hEB cell proliferation returns to normal rates upon return to normoxic conditions. Therefore, while hypoxia effectively stimulates genetic programs in hESCs, optimal culture of hESC-derived cells in hypoxic conditions requires a balance of cell proliferation, differentiation and apoptosis.

Key Words: Human Embryonic Stem Cell, hypoxia, hematopoiesis, Hypoxic Inducible Factor (HIF), embryoid body, differentiation